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Abstract:

The present invention relates to a method for determining the degree of
risk of onset of autism, comprising the step of measuring the
triglyceride concentration or the cholesterol concentration in a very
low-density lipoprotein fraction of plasma or serum isolated from a
subject, or the triglyceride concentration or the cholesterol
concentration of plasma or serum. In addition, the present invention
provides a kit for determining the degree of risk of onset of autism and
a method for screening for a candidate substance for agents for treating
autism using a non-human mammal, in which the above described method is
utilized.

Claims:

1. A method for determining the degree of risk of onset of autism,
comprising the step of measuring a very low-density lipoprotein contained
in plasma or serum isolated from a subject, wherein the degree of risk of
onset of autism of the subject is determined to be high when the
triglyceride concentration in a very low-density lipoprotein fraction of
the plasma or serum is 60 mg/dl or less.

2. The method for determining the degree of risk of onset of autism
according to claim 1, wherein the degree of risk of onset of autism of
the subject is determined to be high when the triglyceride concentration
in a very low-density lipoprotein fraction of the plasma or serum from a
subject who is 18 years old or younger is 60 mg/dl or less.

3. The method for determining the degree of risk of onset of autism
according to claim 1, wherein the degree of risk of onset of autism of
the subject is determined to be high when the triglyceride concentration
in a very low-density lipoprotein fraction of the plasma or serum from a
subject who is 8 years old or younger is 30 mg/dl or less.

4. The method according to claim 1 wherein the autism is high-functioning
autism.

5. A kit for determining the degree of risk of onset of autism,
comprising one or more enzymes selected from the group consisting of
lipoprotein lipase, glycerol kinase, glycerol-3-phosphate oxidase,
peroxidase, ascorbate oxidase, pyruvate kinase, and lactate
dehydrogenase; and a coloring agent.

6. The kit according to claim 5, wherein the autism is high-functioning
autism.

7. A method for screening for a candidate substance for agents for
treating autism, comprising the steps of: (a) administering a test
substance to a non-human mammal; (b) isolating plasma or serum from the
non-human mammal; (c) measuring the triglyceride concentration in a very
low-density lipoprotein fraction of the isolated plasma or serum; and (d)
determining that the test substance is a candidate substance for agents
for treating autism, if indicated by the triglyceride concentration in
the very low-density lipoprotein fraction of the plasma or serum being
increased, as compared with that before the administration of the test
substance.

8. The method for screening according to claim 7, wherein the autism is
high-functioning autism.

9. A method for determining the degree of risk of onset of autism,
comprising the step of measuring a very low-density lipoprotein contained
in plasma or serum isolated from a subject, wherein the degree of risk of
onset of autism of the subject is determined to be high when the
cholesterol concentration in a very low-density lipoprotein fraction of
the plasma or serum is lower than a standard value minus standard
deviation of cholesterol concentrations in very low-density lipoprotein
fractions of plasma or serum of healthy individuals.

10. The method according to claim 9, wherein the autism is
high-functioning autism.

11. A method for determining the degree of risk of onset of autism,
comprising the step of measuring the cholesterol concentration and/or the
triglyceride concentration in plasma or serum isolated from a subject,
wherein the degree of risk of onset of autism of the subject is
determined to be high when the measured concentration of cholesterol is
lower than a standard value minus standard deviation of cholesterol
concentrations in healthy individuals, and/or when the measured
concentration of triglyceride is lower than a standard value minus
standard deviation of triglyceride concentrations in healthy individuals.

12. The method according to claim 11, wherein the autism is
high-functioning autism.

13. A kit for determining the degree of risk of onset of autism,
comprising cholesterol oxidase, peroxidase, and a coloring agent.

14. The kit according to claim 13, wherein the autism is high-functioning
autism.

Description:

TECHNICAL FIELD

[0001] The present application claims priority from Japanese Patent
Application No. 2009-236976 (filed on Oct. 14, 2009); the disclosure of
which is hereby incorporated by reference.

[0002] The present invention relates to a method for discovering an
autistic patient at an early stage. More specifically, the present
invention relates to a method for determining the degree of risk of onset
of autism, comprising the step of measuring a very low-density
lipoprotein contained in plasma or serum isolated from a subject.
Moreover, it relates to a method for determining the degree of risk of
onset of autism, comprising the step of measuring the cholesterol
concentration and/or the triglyceride concentration in plasma or serum.

BACKGROUND ART

[0003] Autism is a disorder that has been first identified as "autistic
disturbances of affective contact" by Dr. Kanner in U.S.A. in 1943. Onset
of autism appears in an extremely early stage (before turning about three
years old), and it includes, as principal symptoms, severe and sustained
impairment in social interaction, deviance in communication, and patterns
of behavior and interest that are restricted or stereotyped. Autism is a
developmental disorder in which the condition changes in the course of
development, and it is assumed to be a yet unspecified high-level central
nervous system disorder.

[0004] High-functioning autism means autism, which does not have mental
retardation. With regard to autism morbidity, it has been reported that 7
to 16 out of 10,000 children suffer from autism, and that
high-functioning autism is about 11% to 34% of the autism (Macintosh et
al., Journal of Child Psychology and Psychiatry 2004).

[0005] The cause of autism has not been clarified at present, and thus,
the radical treatment method for autism has not been developed yet.
Accordingly, educational intervention by experts (which is referred to as
"intensive intervention") based on early detection is important for the
adaptation to social life of autistic patients. The earlier the time of
initiation of such intensive intervention, the more effective it becomes.
It is desired to begin such intervention before or after a child is two
years old, at which the autistic symptoms appear for the first time.
However, since objective and biological criteria for autism have not been
established yet, doctor must diagnose autism only based on symptoms. For
such diagnosis, abundant experience at the site of child psychiatry is
required. Thus, under the circumstances in which there is a shortage of
experts in this field, it is extremely difficult to detect an autistic
patient at an early stage.

[0006] At present, the diagnosis of autism is made by conducting an
interview with subjects using the diagnostic criteria of the American
Psychiatric Association (DSM-IV), ICD-10 of World Health Organization
(WHO), and ADI-R (Autism Diagnostic Interview-Revised).

[0007] Although the cause of autism has not been clarified yet, various
reports have been made so far. For example, enlarged head circumference
is observed at an early stage after the birth of an autistic child, and
thus, it is considered that an increase in neurogenesis and/or
gliogenesis or a decrease in cell death during this period is associated
with the development of autism (McCaffery et al., Progress in
Neurobiology 2005). Other than this report, Fatemi et al. have focused on
developmental disorders of the central nerve system found in Reeler mice,
and have reported that an increase in the mRNA level of a very
low-density lipoprotein receptor (VLDL receptor) that is a receptor of
the responsible gene Reelin of the Reeler mouse is found in the frontal
lobe and cerebellum of an autistic patient after death (Fatemi et al.,
2005). On the other hand, Sharp et al. have made a proteome analysis, and
as a result, they have found a decrease in the concentration of
apolipoprotein B-100 specific to autistic children (Corbett et al.,
2007). Basically, VLDL is a complex of protein and lipid containing the
apolipoprotein B-100, and a change in the expression of this molecule in
vivo is anticipated to be associated with lipid metabolism. However, to
date, there have been no reports of the lipid fractions of peripheral
blood of autistic patients. Also, there have been no reports stating the
relationship between autistic patients and serum lipid levels.

[0008] In addition, there has been disclosed an invention relating to the
diagnosis of autism by measuring the amounts of specific growth factors
in a serum sample (Japanese Patent Laid-Open No. 2008-32447). However,
the behavior of these growth factors is never associated with lipid
metabolism.

[0009] As described above, taking into consideration the specificity of
the pathologic condition of autism, studies have been conducted directed
towards establishing a comprehensive treatment system including
prevention of this disorder based on early diagnosis, early treatment,
and support of patients for social rehabilitation. However, a biological
marker for autism, which enables early diagnosis, has not been developed
yet.

[0015] It is an object of the present invention to provide an objective
and simple method for determining the degree of risk of onset of autism,
using a biological marker, and to enable to provide an appropriate
treatment to the autistic patient at an early stage by finding an
autistic patient at an early stage using the aforementioned method.

Solution to Problem

[0016] In order to achieve the aforementioned object, the present
inventors have collected serum samples from a high-functioning autism
group and a healthy control group and have made a comparison between
them. As a result, the inventors have found that the triglyceride
concentrations in the very low-density lipoprotein fractions of underage
autistic patients are significantly decreased, and based on such a
phenomenon, they have completed the present invention.

[0017] Moreover, the present inventors have collected serum samples from a
high-functioning autism group and a healthy control group and have made a
comparison between them. As a result, the inventors have found that the
total amounts of cholesterol and triglyceride are significantly decreased
in underage autistic patients, and that the cholesterol concentrations in
the very low-density lipoprotein fractions of underage autistic patients
are significantly decreased. The inventors have completed the present
invention based on these phenomena.

[0018] Specifically, the present invention relates to a method for
determining the degree of risk of onset of autism, comprising the step of
measuring a very low-density lipoprotein contained in plasma or serum
isolated from a subject, wherein the degree of risk of onset of autism of
the subject is determined to be high when the triglyceride concentration
in a very low-density lipoprotein fraction of the plasma or serum is 60
mg/dl or less.

[0019] The above described subject is desirably 18 years old or younger.

[0020] In addition, when the above described subject is 8 years old or
younger and the triglyceride concentration in the very low-density
lipoprotein fraction of the serum is 30 mg/dl or less, the degree of risk
of onset of autism of the subject is determined to be high.

[0021] Moreover, the present invention provides a kit for determining the
degree of risk of onset of autism, comprising one or more enzymes
selected from the group consisting of lipoprotein lipase, glycerol
kinase, glycerol-3-phosphate oxidase, peroxidase, ascorbate oxidase,
pyruvate kinase, and lactate dehydrogenase; and a coloring agent.

[0022] Furthermore, the present invention provides a method for screening
for a candidate substance for agents for treating autism, comprising the
steps of: [0023] (a) administering a test substance to a non-human
mammal; [0024] (b) isolating plasma or serum from the non-human mammal;
[0025] (c) measuring the triglyceride concentration in a very low-density
lipoprotein fraction of the isolated plasma or serum; and [0026] (d)
determining that the test substance is a candidate substance for agents
for treating autism, if indicated by the triglyceride concentration in
the very low-density lipoprotein fraction of the plasma or serum being
increased, as compared with that before the administration of the test
substance.

[0027] Further, the present invention provides a method for determining
the degree of risk of onset of autism, comprising the step of measuring a
very low-density lipoprotein contained in plasma or serum isolated from a
subject, wherein the degree of risk of onset of autism of the subject is
determined to be high when the cholesterol concentration in a very
low-density lipoprotein fraction of the plasma or serum is lower than a
standard value minus standard deviation of the cholesterol concentrations
in a very low-density lipoprotein fractions of the plasma or serum of
healthy individuals.

[0028] Still further, the present invention provides a method for
determining the degree of risk of onset of autism, comprising the step of
measuring the cholesterol concentration and/or the triglyceride
concentration in plasma or serum isolated from a subject, wherein the
degree of risk of onset of autism of the subject is determined to be high
when the measured concentration of cholesterol is lower than a standard
value minus standard deviation of the cholesterol concentrations in
healthy individuals, and/or when the measured concentration of
triglyceride is lower than a standard value minus standard deviation of
the triglyceride concentrations in healthy individuals.

[0029] Still further, the present invention provides a kit for determining
the degree of risk of onset of autism, comprising cholesterol oxidase,
peroxidase, and a coloring agent.

[0030] The above described autism may be high-functioning autism.

Advantageous Effects of Invention

[0031] According to the present invention, biological criteria for
determining the degree of risk of onset of high-functioning autism have
been determined for the first time. In addition, the present invention
can be carried out by a method involving collection of peripheral blood,
which is simple and which is able to reduce burden to subjects to the
minimum.

[0032] According to the present invention, the diagnosis of autism, which
has mainly been made by a doctor's subjective view so far, can be made by
a comprehensive diagnosis attended with biological criteria. Thus, the
improvement of a diagnostic technique can be anticipated. The earlier the
intensive intervention that is given to autistic patients, the more
effective it would be. Since the method of the present invention can be
applied to infants and children, it enables early finding of autism and
initiation of intensive intervention to autistic children at an early
stage. This is extremely effective for autistic children who will
organize their social life in the future. Moreover, nowadays, troubles
caused by autistic patients have gained prominent attention. From the
viewpoint of coping with these problems, the present invention has great
social effects.

[0033] The present invention enables simple and reliable diagnosis of
autism at an early stage. In particular, by measuring a triglyceride
concentration in a VLDL fraction of a human blood sample at two stages,
the diagnosis of autism can be easily assisted.

BRIEF DESCRIPTION OF DRAWINGS

[0034] FIG. 1 shows the mean values of lipoprotein fraction concentrations
in a serum lipid in an underage high-functioning autistic patient group
and an underage healthy control group.

[0035] FIG. 2 shows the relationship between the cholesterol concentration
in a VLDL fraction and age, in autistic patients and healthy control
subjects.

[0036] FIG. 3 shows the relationship between the triglyceride
concentration in a VLDL fraction and age, in autistic patients and
healthy control subjects, and a cutoff value used to determine
high-functioning autistic patients.

DESCRIPTION OF EMBODIMENTS

[0037] The present invention relates to a method for detecting an autistic
patient at an early stage. More specifically, the present invention
relates to a method for determining the degree of risk of onset of
autism, comprising the step of measuring a very low-density lipoprotein
contained in serum isolated from a subject.

1. Definition

[0038] "Autism" is one of pervasive developmental disorders, which is
diagnosed during childhood. Main symptoms, such as communication
disorder, impaired social interaction, restricted interest and behavior,
hyperaesthesia, and hyperactive tendency, appear before an autistic
patient turns 3 years old. "High-functioning autism" means the above
described autism, which does not have mental retardation.

[0039] In the invention of the present application, the "degree of risk of
onset of autism" means a criterion for determination of autism, which is
used for determining whether or not a subject will develop autism, or
whether or not a subject has developed autism. The higher the degree of
risk of onset of autism, the more likely the subject will develop autism,
or the subject is diagnosed to be an autistic patient. In contrast, the
lower the degree of risk of onset of autism, the more unlikely the
subject will develop autism, or it is determined that the subject is
healthy.

[0040] "Very low-density lipoprotein (VLDL)" is one type of lipoprotein
present in blood. Such lipoproteins are classified depending on the
density measured by centrifugation. The lipoprotein includes a
chylomicron (CM), an intermediate-density lipoprotein (IDL), a
low-density lipoprotein (LDL) and a high-density lipoprotein (HDL), as
well as VLDL. These lipoproteins share the property that they are
composed of protein, triglyceride, free cholesterol, cholesterol ester,
and phospholipid. However, they are different from one another in that
each lipoprotein comprises a characteristic apoprotein and has a
different lipid composition.

[0041] A "very low-density lipoprotein fraction" or "VLDL fraction" means
a fraction having a density of 0.950 to 1.006 g/ml, which is obtained by
centrifugation of plasma or serum. Likewise, a "chylomicron fraction" or
"CM fraction" means a fraction having a density of <0.95 g/ml, which
is obtained by centrifugation of plasma or serum. A "low-density
lipoprotein fraction" or "LDL fraction" means a fraction having a density
of 1.019 to 1.063 g/ml, which is obtained by centrifugation of plasma or
serum. A "high-density lipoprotein fraction" or "HDL fraction" means a
fraction having a density of 1.063 to 1.210 g/ml, which is obtained by
centrifugation of plasma or serum.

[0043] The term "underage" or "child" is used in the present invention to
mean a human who is 18 years old or younger.

[0044] The term "cutoff value" is used herein to mean a value used to
distinguish a target disease group from a non-disease group, when
focusing on a certain substance. When such a target disease group is
distinguished from a non-disease group, if the value is smaller than the
cutoff value, it can be determined to be negative, and if the value is
greater than the cutoff value, it can be determined to be positive. On
the other hand, if the value is smaller than the cutoff value, it can be
determined to be positive, and if the value is greater than the cutoff
value, it can be determined to be negative, thereby determining the
development of the disease.

[0045] Indicators used for the purpose of evaluating the clinical
usefulness of a cutoff value include sensitivity and specificity.

[0046] A certain population is determined using a cutoff value. Among
suspected patients of a certain disease, patients who are determined to
be positive are defined as "a" (true positive), patients who are
determined to be negative although they are affected with the certain
disease are defined as "b" (false-negative), patients who are determined
to be positive although they are not affected with the certain disease
are defined as "c" (false-positive), and patients who are determined to
be negative and they are not affected with the certain disease are
defined as "d" (true negative). The value represented by a/(a+b)
indicates sensitivity (true positive rate), and the value represented by
d/(c+d) indicates specificity (true negative rate).

[0047] With regard to distribution of the measurement values of a target
disease group and a non-disease group, the distribution is generally
overlapped. Accordingly, sensitivity and specificity are changed by
increasing or decreasing a cutoff value. When the cutoff value is
decreased, sensitivity is increased, but specificity is decreased. In
contrast, when the cutoff value is increased, sensitivity is decreased,
but specificity is increased. For a determination method, both the values
of sensitivity and specificity are preferably high. In addition, a
determination method, in which the values of sensitivity and specificity
do not exceed 0.5, is not considered to be useful.

2. Method for Determining the Degree of Risk of Onset of Autism

[0048] The present invention relates to a method for determining the
degree of risk of onset of autism, comprising the step of measuring a
very low-density lipoprotein contained in plasma or serum isolated from a
subject.

(1) Isolation Step

[0049] As a sample to be measured in the present invention, in general,
plasma or serum is prepared from blood collected from a subject, and is
then used. In the present invention, peripheral blood can be used as a
sample. As a method of preparing plasma or serum, a method well known in
the art can be used. The prepared plasma or serum may be frozen for
preservation before subjecting to measurement.

(2) Measurement Step

[0050] In the present invention, a measurement step is carried out by
separating plasma or serum isolated from a subject to obtain fractions
according to a method well known in the art, such as an ultracentrifugal
method or gel filtration, and then measuring the triglyceride
concentration or cholesterol concentration in each fraction. If a gel
filtration HPLC method is applied, serum can be separated, and each
fraction can be then detected and quantified. There is no difference in
the lipid concentration between in plasma and in serum.

[0051] As described above, depending on density, lipoproteins contained in
plasma or serum are divided into a chylomicron, IDL, VLDL, LDL, and HDL.
Separation of serum into individual fractions and the subsequent
quantification can be carried out according to a well known method such
as a gel filtration HPLC method.

(i) Measurement of Triglyceride Concentration

[0052] In the present invention, the triglyceride concentration in VLDL
may be directly measured, and the degree of risk of onset of autism may
be then determined based thereon. Alternatively, a two-stage measurement
may be carried out. Specifically, the total amount of triglyceride
contained in plasma or serum may be first measured. Then, when the total
amount of triglyceride is less than a predetermined value, the
triglyceride concentration in a VLDL fraction may be measured, and the
degree of risk of onset of autism may be then determined. In the case of
carrying out such a two-stage measurement, it is preferable that the
triglyceride concentration in a VLDL fraction be measured to determine
the degree of risk of onset of autism, when the total amount of
triglyceride is less than 80 mg/dl, preferably less than 70 mg/dl, and
more preferably less than 60 mg/dl. By performing the measurement at two
stages, the diagnosis of autism can be easily assisted.

[0053] The concentration of triglyceride can be determined by measuring it
by a glycerol kinase (GK)-glycerol-3-phosphate oxidase (GPO) method, a
glycerol oxidase (GOD) method, a glycerol dehydrogenase (GDH) method,
etc., based on a method of measuring glycerin produced from triglyceride
with the use of lipoprotein lipase (LPL).

[0054] For instance, in the case of a GPO-DAOS method, the amount of
triglyceride can be measured by quantifying hydrogen peroxide produced
with the use of lipoprotein lipase (LPL), glycerol kinase (GK),
glycerol-3-phosphate oxidase (GPO) and peroxidase (POD), using DAOS.
Specifically, the GPO-DAOS method comprises the following steps: [0055]
1) degrading triglyceride contained in a sample into glycerin and fatty
acid by the action of lipoprotein lipase (LPL); [0056] 2) converting the
produced glycerin to glycerol-3-phosphate by the action of glycerol
kinase (GK) in the presence of ATP; [0057] 3) oxidizing the produced
glycerol-3-phosphate by the action of glycerol-3-phosphate oxidase (GPO),
and at the same time, producing hydrogen peroxide; [0058] 4) allowing the
produced hydrogen peroxide to cause quantitative oxidative condensation
of N-methyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt
(DAOS) and 4-aminoantipyrine by the action of peroxidase (POD), so as to
produce a blue dye; and [0059] 5) measuring the absorbance of the
produced blue dye, thereby calculating the triglyceride concentration in
the sample.

[0060] Likewise, in the case of a "NAD" method, the produced hydrogen
peroxide is quantified using lipoprotein lipase (LPL), glycerol kinase
(GK), pyruvate kinase, and lactate dehydrogenase.

(ii) Measurement of Cholesterol Concentration

[0061] In the present invention, the cholesterol concentration in VLDL may
be directly measured, and the degree of risk of onset of autism may be
then determined based thereon. Alternatively, a two-stage measurement may
be carried out. Specifically, the total amount of cholesterol contained
in plasma or serum may be first measured. Then, when the total amount of
cholesterol is less than a predetermined value, the cholesterol
concentration in a VLDL fraction may be measured, and the degree of risk
of onset of autism may be then determined. By performing the measurement
at two stages, the diagnosis of autism can be easily assisted.

[0062] The concentration of cholesterol can be determined by measuring it
by a cholesterol oxidase-DAOS method or the like.

[0063] For instance, in the case of the cholesterol oxidase-DAOS method,
the amount of cholesterol can be measured by quantifying hydrogen
peroxide produced with the use of cholesterol oxidase, using DAOS.
Specifically, the cholesterol oxidase-DAOS method comprises the following
steps: [0064] 1) oxidizing cholesterol contained in a sample by the
action of cholesterol oxidase, so as to produce a ketone body and
hydrogen peroxide; [0065] 2) allowing the produced hydrogen peroxide to
cause quantitative oxidative condensation of
N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt
(DAOS) and 4-aminoantipyrine by the action of peroxidase, so as to
produce a blue dye; and [0066] 3) measuring the absorbance of the
produced blue dye, thereby calculating the cholesterol concentration in
the sample.

[0067] In another aspect of the present invention, the measurement step is
carried out by measuring the total amount of triglyceride or the total
amount of cholesterol contained in plasma or serum isolated from a
subject.

(3) Determination Step

[0068] The present inventors have found that the triglyceride
concentration in a VLDL fraction is almost constant in healthy
individuals regardless of their age, but that, in the case of autistic
patients, the triglyceride concentration in a VLDL fraction is low in
their childhood and then increases with aging.

[0069] Based on this finding, in the present invention, when the
triglyceride concentration in the VLDL fraction of a subject, which is
measured by the above described method, is lower than a standard value
minus standard deviation of the triglyceride concentrations in the VLDL
fractions of healthy individuals, the degree of risk of onset of autism
of the subject is determined to be high. Specifically, when the
triglyceride concentration in the VLDL fraction of a subject is 60 mg/dl
or less, and preferably 50 mg/dl or less, the degree of risk of onset of
autism of the subject is determined to be high. Preferably, when the
triglyceride concentration in the VLDL fraction of a subject who is 18
years old or younger is 50 mg/dl or less and preferably 40 mg/dl or less,
the degree of risk of onset of autism of the subject is determined to be
high. More preferably, when the triglyceride concentration in the VLDL
fraction of a subject who is 8 years old or younger is 40 mg/dl or less
and preferably 30 mg/dl or less, the degree of risk of onset of autism of
the subject is determined to be high.

[0070] Moreover, the present inventors have found that the cholesterol
concentration in the very low-density lipoprotein fraction of an autistic
patient is significantly decreased, when compared with those of healthy
individuals. Based on this finding, in the present invention, when the
cholesterol concentration in the VLDL fraction of a subject, which is
measured by the above described method, is lower than a standard value
minus standard deviation of the cholesterol concentrations in the VLDL
fractions of healthy individuals, the degree of risk of onset of autism
of the subject is determined to be high. Specifically, when the
cholesterol concentration in the VLDL fraction of a subject is 20 mg/dl
or less, and preferably 15 mg/dl or less, the degree of risk of onset of
autism of the subject is determined to be high.

[0071] Furthermore, the present inventors have found that the total
amounts of cholesterol and triglyceride of autistic patients are
significantly decreased, when compared with those of healthy individuals.
Based on this finding, in the present invention, when the triglyceride
concentration or cholesterol concentration of the plasma or serum of a
subject, which is measured by the above described method, is lower than a
standard value minus standard deviation of the triglyceride
concentrations or cholesterol concentrations of healthy individuals, the
degree of risk of onset of autism of the subject is determined to be
high. Specifically, when the cholesterol concentration is 160 mg/dl or
less and preferably 150 mg/dl or less, or when the triglyceride
concentration is 80 mg/dl or less and preferably 60 mg/dl or less, the
degree of risk of onset of autism of the subject is determined to be
high.

[0072] According to the present invention, the degree of risk of onset of
high-functioning autism can be determined by a simple method using
peripheral blood, at a high proper diagnosis rate in an early stage.

3. Kit for Determining the Degree of Risk of Onset of Autism

[0073] The kit according to the present invention may be a kit for
carrying out the method for determining the degree of risk of onset of
autism, described in the above section 2. Specific constitution,
materials, devices and the like included in the kit are not particularly
limited.

[0074] For example, the kit of the present invention comprises, but not
limited to, reagents for measuring the triglyceride in isolated
peripheral blood plasma or peripheral blood serum, or in the VLDL of
peripheral blood: one or more enzymes which are selected from lipoprotein
lipase (LPL), glycerol kinase (GK), glycerol-3-phosphate oxidase (GPO),
glycerol oxidase (GOD), glycerol dehydrogenase (GDH), pyruvate kinase,
lactate dehydrogenase, peroxidase, and the like, and a coloring agent
such as DAOS or FDAOS. The present kit may further comprise a buffer
necessary for the measurement.

[0075] In the case of measuring the triglyceride concentration according
to the GPO-DAOS method for example, the kit of the present invention may
comprise one or more selected from glycerin, a buffer (PIPES; pH 6.5),
lipoprotein lipase, adenosine-5'-phosphate disodium trihydrate (ATP),
glycerol kinase, glycerol-3-phosphate oxidase (GPO), peroxidase,
N-ethyl-N-(2'-hydroxy-3'-sulfopropyl)-3,5-dimethoxyaniline sodium (DAOS),
4-aminoantipyrine and ascorbate oxidase.

[0076] Further, the kit of the present invention may comprise, but not
limited to, reagents for measuring the cholesterol in isolated peripheral
blood plasma or peripheral blood serum, or in the VLDL of peripheral
blood, such as cholesterol oxidase, peroxidase, and a coloring reagent.

[0077] Using the above described kit, the degree of risk of onset of
high-functioning autism can be determined.

[0078] Still further, the kit for determining the degree of risk of onset
of autism according to the present invention can also be used as a kit
for the after-mentioned method for screening for a candidate substance
for agents for treating autism.

4. Method for Screening for Candidate Substance for Agents for Treating
Autism

[0079] The determination method or determination kit of the present
invention can be applied to a method for screening for a candidate
substance for agents for treating autism using a non-human mammal. As
such a non-human mammal, mammals other than humans may be used. Examples
of such a non-human mammal include a mouse, rat, and monkey.

[0080] The present invention provides a method for screening for a
candidate substance for agents for treating autism, comprising the steps
of: [0081] (a) administering a test substance to a non-human mammal;
[0082] (b) isolating plasma or serum from the non-human mammal; [0083]
(c) measuring the triglyceride concentration in a very low-density
lipoprotein fraction of the isolated plasma or serum; and [0084] (d)
determining that the test substance is a candidate substance for agents
for treating autism, if indicated by the triglyceride concentration in
the very low-density lipoprotein fraction of the plasma or serum being
increased, as compared with that before the administration of the test
substance.

[0085] In another aspect of the present invention, increases in the
triglyceride concentration or cholesterol concentration in peripheral
blood plasma or peripheral blood serum, or the cholesterol concentration
in a very low-density lipoprotein fraction of peripheral blood plasma or
peripheral blood serum can be used as an indicator for screening for a
candidate substance for agents for treating autism.

[0086] Using this screening method, a candidate substance for agents for
treating high-functioning autism can be selected.

[0087] Hereinafter, the present invention will be described more in detail
in the following examples. However, these examples are not intended to
limit the scope of the present invention.

EXAMPLES

1. Subject

[0088] As subjects, 106 male patients with high-functioning autism
[average age: 14.7 years old (standard deviation: 5.9); age range: 6 to
30 years old] were selected. At the same time, 112 healthy individuals in
the same age range [average age: 15.1 years old (standard deviation:
6.2); age range: 6 to 30 years old] were selected as healthy controls.
All patients were diagnosed in accordance with diagnostic criteria for
high-functioning autism, ADI-R (Autism Diagnostic Interview-Revised).

2. Experimental Method

[0089] A serum specimen was collected from each subject, and it was then
preserved at -80° C. before subjecting to measurement. The
cholesterol and triglyceride in a lipid fraction were subjected to a
measurement comparison according to a HPLC method. The triglyceride
concentration in a VLDL fraction was measured according to a gel
filtration HPLC method, and this measurement was performed by
Skylight-Biotech on a commission basis.

3. Statistical Analysis

[0090] Data were shown using mean values. Statistical analysis between the
two groups was carried out by a t-test. A p-value of less than 0.01 was
defined to be statistically significant.

4. Results

(1) Decrease in Serum Lipid Concentration in Autistic Patients

[0091] The mean values of serum lipid concentrations in lipoprotein
fractions, in underage healthy controls and underage subjects with
high-functioning autism, are shown in Table 1 and FIG. 1.

[0092] As is apparent from FIG. 1, it was found that, with regard to the
serum lipids of the underage subjects with high-functioning autism, the
total amounts of both cholesterol and triglyceride were significantly
decreased, as compared with the underage healthy controls. Moreover, as a
result of the measurement of the cholesterol and triglyceride
concentrations in each lipoprotein fraction, chylomicron, VLDL, LDL and
HDL, it was also found that both the cholesterol and triglyceride
concentrations were decreased in the VLDL fraction and the HDL fraction,
and that the concentrations were particularly significantly decreased in
the VLDL fraction. There was found no significant difference in the
chylomicron fraction and the LDL fraction.

(2) Correlation Between Decrease in Cholesterol Concentration/Triglyceride
Concentration in VLDL Fraction and Age

[0093] The relationship between the cholesterol concentration in a VLDL
fraction and age, in autistic patients and healthy controls, is shown in
FIG. 2. The relationship between the triglyceride concentration in a VLDL
fraction and age, in autistic patients and healthy controls, is shown in
FIG. 3.

[0094] As is apparent from FIG. 2, the cholesterol concentrations in the
VLDL fractions of healthy controls are constant regardless of age. On the
other hand, the cholesterol concentrations in the VLDL fraction of
autistic patients are the lowest in the childhood, and then they increase
with aging.

[0095] Since triglyceride is a major lipid contained in VLDL, the
aforementioned tendency was much clearly observed in the case of
triglyceride concentration (FIG. 3). Accordingly, a decrease in the
triglyceride concentration in a VLDL fraction was considered to be
effective for early diagnosis of high-functioning autism.

(3) Cutoff Value

[0096] The tendency that the concentrations in the VLDL fractions of
autistic patients are low in the childhood of autistic patients and then
increase with aging was more clearly observed in the case of the
triglyceride concentration than in the case of the cholesterol
concentration. Thus, the cutoff value of the triglyceride concentration
was investigated.

[0097] As a result, when the triglyceride concentration in the VLDL
fraction of patients who were 8 years old or younger was lower than 30
mg/dl, patients with high-functioning autism could be selected at high
rates (sensitivity: 83%; specificity: 90%; proper diagnosis rate: 86%)
(FIG. 3). It is to be noted that sensitivity was 63%, specificity was
72%, and proper diagnosis rate was 67% in the case of all of underage
patients, namely, patients who were 18 years old or younger.

[0098] Therefore, as a result of the present experiment, it could be
confirmed that the triglyceride concentration in a VLDL fraction of blood
is useful as a biological marker for autism.

INDUSTRIAL APPLICABILITY

[0099] According to the present invention, the diagnosis of autism, which
has mainly been made by a doctor's subjective view so far, can be carried
out by introduction of biological criteria. Accordingly, the improvement
of a diagnostic technique can be anticipated. In addition, the earlier
the time of initiation of intensive intervention given to autistic
patients, the more effective that can be anticipated. The method of the
present invention can be applied to infants or children, and thus, it
enables the establishment of a comprehensive treatment system including
prevention of this disease, early treatment, and support of patients for
social rehabilitation, based on early diagnosis.